DESCRIPTION: The relation of RNA structure and function will be studied in a small catalytic RNA (the hammerhead ribozyme) and a sequence specific RNA-protein interaction (MS2 coat protein bound to a small hairpin). Both projects were chosen because the RNA molecules and the protein are relatively small, the biology is well understood, and accurate biochemical assays could be developed. Efforts of this laboratory and others in the past ten years have resulted in these two systems reaching a stage of development such that they are often considered "model" systems where concepts of RNA catalysis and RNa-protein interactions can be refined and new methods to study RNa can be developed. The recent availability of X-ray crystal structures in both systems form the basis of the next stage of analysis. Dr. Uhlenceck will make use of the excellent synthetic control of RNA and quantitative biochemical and biophysical methods to carry out detailed structure-function experiments, with the ultimate goals of understanding the mechanism of hammerhead catalysis and how the high specificity of MS2 coat protein binding to RNA is achieved. The three hammerhead specific aims involve introducing atom or functional group modifications into specific sites in kinetically well-characterized hammerheads. Aims are to understand how structure must rearrange to reach the transition state, identify candidates for future structural studies, and develop ideas of how divalent metal ions participate in the reaction. The two MS2 aims involve making conservative protein and nucleic acid mutations to obtain a thermodynamic description of the specific RNA-protein interaction. The specificity of the system will be explored with in vitro selection experiments and "swap" experiments with the related Qbeta system.